Publications and other reference materials referred to herein are incorporated herein in their entireties by this reference.
Nucleic acid hybridization assays have emerged as an important method for the clinical detection of viral and microbial pathogens and genetic disorders. Assay formats are predominantly based on heterogeneous hybridization in which the target nucleic acid is sequestered on a solid support to allow separation of hybridized and unhybridized detection probe (Matthews and Kricka, Anal. Biochem. 169:1-25 (1988)). While these formats display good sensitivity, the necessity of separation and wash steps makes such assays time-consuming and also makes automation difficult.
Thus, presently available heterogeneous methods for detecting or quantitating nucleic acid sequences by hybridization generally involve a large number of steps and are laborious to perform. Typically, a DNA sequence to be assayed is denatured and bound to a solid support and then incubated with a probe complementary to the target nucleic acid (or a portion thereof). The probe is labeled with, for example, a radioactive label, a chemiluminescent label or an enzyme, such as alkaline phosphatase. After incubation and washing of the solid phase, it is then normally developed to give a signal, for example, in the case of a radioactive label, by autoradiography.
Sandwich-type assays are also used, in which a so-called "capture" nucleic acid probe having a sequence complementary to the target sequence is bound to a solid support, and then allowed to hybridize to the target sequence. A labeled probe is then allowed to hybridize to another portion of the target sequence. After washing, the label is detected. A "sandwich" may also be formed in solution with two probes, one containing a label and the other containing one half of an affinity pair. After hybridization, the solution is contacted with a solid phase to which the other half of the affinity pair is bound. After washing, the signal of the label is detected. For descriptions of various hybridization methods, see, for example, Hames and Higgins, eds., Hybridization, IRL Press (1985) and Matthews and Kricka, supra.
More recently, a number of homogeneous hybridization assays have been reported which exploit the properties of donor and acceptor fluorophor-labeled oligonucleotides to transfer or quench fluorescence energy when hybridized to an analyte (Morrison et al., Anal. Biochem. 183:231-244 (1989); Cardullo et al., Proc. Natl. Acad. Sci. USA 85:8790-8794 (1988)). Arnold et al., Clin. Chem. 35:1588-1594 (1989), have described a chemiluminescence-based detection assay which exploits the differential rates of hydrolysis of acridinium ester-labeled oligonucleotide probes. While sensitivities in the attomole range are reported, their assay formats require the addition of reagents to trigger the chemiluminescent reactions.
Homogeneous assays permit the monitoring of hybridization reactions in real time. However, homogeneous assays which use conventional fluorophors, such as fluorescein, are compromised by background fluorescence from biological test samples. Real time methods enable the measurement of the reaction kinetics so that the assay conditions can be optimized to quantitate the particular amount of the oligonucleotide of interest. Thus, homogeneous assays are useful to measure, for example, viral load or bacterial load, and to monitor the course of various disease states. Real-time monitoring of hybridization procedures reduces steps involved, as well as manipulation of samples, which results in lower contamination, as well as ease in automation.
Nucleic acid amplification methods, such as polymerase chain reaction (PCR), generate nucleic acid sequences which are then assayed. Thus, nucleic acid amplification methods incorporate hybridization steps, as well.
In PCR, a pair of primers (one primary and one secondary) is employed in excess to hybridize at the outside ends of complementary strands of the target nucleic acid. The primers are each extended by a polymerase using the target nucleic acid as a template. The extension products become target sequences themselves, following dissociation from the original target strand. New primers are then hybridized and extended by a polymerase, and the cycle is repeated to increase geometrically the number of target sequence molecules. PCR is described in more detail, for example, in U.S. Pat. Nos. 4,683,195 and 4,683,202. See also, Saiki et al., Science 230:1350-1354 (1985); Mullis and Faloona, Methods Enzymol. 155:335-350 (1987); Eisenstein, New Engl. J. Med. 322:178-183 (1990). Modifications, adaptions and enhancements of the basic PCR protocol are numerous (See, e.g., Erlich, ed., PCR Technology: Principles and Amplifications for DNA Amplification, Stockton Press, New York (1989); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, New York (1990)).
More recently, an amplification method based on cycles of oligonucleotide-targeted ligation, called ligase chain reaction (LCR), has been described (Barany, PCR Methods and Applications 1:5-16 (1991)). Similar to the PCR protocol, the LCR method includes thermocycling steps to permit the denaturing of newly ligated oligonucleotide duplexes so that these products can serve as templates for subsequent cycles of amplification.
Allele-specific LCR employs four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides which hybridize to the opposite strand. Thermostable DNA ligase will covalently link each set, provided there is sufficient complementarity at the junction. Because the oligonucleotide products from one round may serve as substrates during the next round, the signal is amplified exponentially, analogous to PCR amplification. A single-base mismatch at the oligonucleotide junction will not be amplified, and is therefore distinguished. A second set of mutant-specific oligonucleotides is used in a separate reaction to detect the mutant allele.
An alternate amplification technique is the transcription-based amplification system (TAS). Each cycle of TAS is composed of two steps. The first step is a cDNA synthesis step that produces one copy of a double-stranded DNA template for each copy of target RNA or DNA target nucleic acid. During the course of the cDNA step, a sequence recognized by a DNA-dependent RNA polymerase is inserted into the cDNA copy of the target sequence to be amplified. The second step is the amplification of the target sequence by the transcription of the cDNA template into multiple copies of RNA. This procedure has been applied to, for example, the detection of human immunodeficiency virus type 1 (HIV-1)-infected cells (Kwoh, et al., Proc. Natl. Acad. Sci. USA 6:1173-1177 (1989)).
These amplification techniques are potentially useful in any situation which requires the examination of DNA. The methods have been used, for example, in research fields involving genetic analysis and other fields, such as archeology and forensic pathology. By amplifying target DNA, amplification methods can supplement or replace many standard cloning methods, such as site-specific mutagenesis, complementary and genomic DNA cloning, analysis of protein-DNA interaction, DNA and RNA sequencing, and gene therapy manipulations.
With regard to specific applications in medicine, amplification methods such as PCR are used to determine whether a given sequence of DNA exists in a particular clinical specimen. Apart from forensic pathology and diagnosis, amplification methods are useful in studying the pathogenesis of disease due to their ability to detect specific DNA sequences that can be correlated with defined pathologic conditions.
From the above discussion, it can be seen that, although many types of nucleic acid hybridization and amplification methods exist, they have several disadvantages.